Bums (1990) 16, (4) 249-253 Printed in Great Britain 249

Roles of thromboxane and its inhibitor anisodamine in burn shock

Huang Yuesheng, Li Ao(Ngao) and Yang Zongcheng Bum Institute, Southwestern Hospital, Third Military Medical College of PLA, Chongqing, China

Thromboxane (TXA,) and prostacyclin (PGI,) levels, circdatory platelet aggregate ratios (CPAR), CPK, LDH, GOT, platelet counts, blood viscosity, cortisol and urine epinephn’ne con&nts were determined in 42 burned patients who were divided into two groups: Group I control (n = 34) and Group II (n = 8) treated with T&4, synthesis inhibitor, anisodamine. It was found that in controls, both 7X4, and the DtA,/PGl, ratio increased significantly. There was no marked differolce in FGI, levels between the lwo groups. Platelet counts and CPAR decreased, while blood viscosity, CPK, LDH, GOT cortisol and epkephn’ne in the controls were all significantly higher than those found in Group II patients. All these findings suggesfed that the changes of I%, and the TxA,/pGI, ratios pkzyed an imporfanf role in the haemody- namics and haemorrheology in burn shock. The TX/I, synthesis inhibitor, anisodamine, showed beneficial effects by restoring fhe haemodynamic and rheological disturbances towards normal by virtue of their ability to induce vascular constriction, platelet aggregation, cellular a%rucfion, destabil- ization of membranes and release of chemical mediafors (including enzymes). Furthermore, at 1-3 days postbum, fhe levels of CPK, LDH and GOT in controls were higher than those m-red at 12 h postbum, but this phenomenon was not marked in the treated group, suggesting that after resusciiation, reperfusion damage had occurred and 7X4, might be responsible for the damage. It is assumed that an&&amine could proteck tissues fram reperfusion damage. The findings also suggested that anisodamine couM quicken the restoration of neuroendocrine disturbance initiated by shock (stress)).

Introduction The treatment of shock is one of the crucial steps in the successful treatment of patients with major bums, but the precise mechanisms which induce burn shock are not yet fully understood. This ignorance may limit the optimal means of treating shocked burned patients. It is thus imperative to try to understand the mechanisms leading to microvascular changes postburn and to lessen or block this change with specific drugs. It has been reported by Alemayehu et aI. (1987), Bittennan et al. (1986) Carmona et aI. (1984) and Reines et aI. (1982) that an imbalance of thromboxane (TXA,) and prostacyclin (PGI,) levels plays an important role in haemorrhagic and septic shock.

0 1990 Butterworth-Heinema Ltd 0305-4179f90/040249-05

In this study, we investigated the changes of TXA, and PGI, and some other relevant variables in burned patients in order to define the roles of TxA,/PGI, imbalance in bum shock and the therapeutic effects of TXA, synthesis inhibitor, anisodamine, in the antishock treatment.

Materials and methods Forty-two patients admitted to the Bum Institute of the Southwestern Hospital, Chongqing from February, 1987 to February, 1988, were studied. Twenty-seven patients were male and 15 were female. The average patient age was 28.9 & 10.0 years (16-58 years). The average percentage of total body burn was 53.5 f 17.8 per cent (30-85 per cent) and average percentage of deep body burn was 13.0 f 15.2 per cent. AI1 42 patients survived.

The 42 patients were randomly divided into two groups. Group 1, the control group (n = 34), had an average age of 28.3 f 10.1 years, an average TBSA of 53.4 &18.7 per cent and an average area of full thickness skin loss of 12.4 f 15.6 per cent. AI1 patients in this group received conventional treatment in our Institute after admission. The volume of intravenous fluid replacement required was estimated according to the formula- developed in our Institute for adults (T&k I; Bum Research Unit, 1977). Blood and urine samples were collected and measured at 12 h, I, 2,3,5 and 7 days postbum. Group II patients (n = 8) had an average age of 31.5 f 9.7 years, and average TBSA of 53.9f 14.8 per cent and an average full thickness skin loss of 17.1 f 13.2 per cent. In addition to the conventional treatment, three doses (each of 0.5 mg/kg body weight diluted in 100 ml of 5 per cent dextrose) of TXA, synthesis inhibitor, anisodamine (Changzheng Pharmaceutical Factory, Suzhou), were infused intravenously within 48 h postbum with an interval of 8.4 f 5.9 h. Each infusion lasted about 30min. Measure- ments were made as in Group I patients.

Radioimmunoassay techniques were adapted to measure TXB,, the stable degradation product of TXA, and 6-keto- PGF,,, the stable degradation product of PGI,, in plasma (RIA kits supplied by the General Hospital of PLA, Beijing) (Li et al., 1985). Circulatory platelet aggregate ratio (CPAR) was determined by the method of Wu and Hoak (1974). Blood viscosity was measured with a Model NZ-4 viscosity

250 BLlms(1990) vol. 16iNo.4

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z. 400 [ 0 h --_i---i-___*___ ;,,;+ 7

0.5 1 2 3 5 7 Days postburn

Figure 1. Changes in TXB,, 6-keto-PGF,, and TXES,/6-keto- PGF,, ratio. -, Anisodamine-treated group (n = 8); -.-‘-.-, control group (n= 34).+P<O.O5; ??'P<O.Ol.

machine (made in Tianjin). Creatinine phosphokinase (CPK), lactic dehydrogenase (LDH), glutamic-oxaloacetic transami- nase (GOT) and platelet counts were determined by routine methods. Twenty-four-hour urine epinephrine and plasma cortisol were determined by fluorophotomehy and radioim- munoassay, respectively.

Statistical analysis All results were expressed as mean values f standard error. Statistical analyses were performed using Student’s t test, analysis of regression and correlation. Results were con- sidered significant if PCO.05 and highly significant if lJ< 0.01.

Results TXBz, 6-keto-PGF,, and TXBJ6-keto-PGF1, ratio

At 12 h postbum there was no significant difference in TXB, between the two groups. Beginning from 24 h up to 3 days postbum the levels of TXB, were markedly higher in controls than in anisodamine-treated patients. TXB,/&keto- PGF,, ratios were also higher at 2,3,5 and 7 days postbum in the control group than in the treated group. However the 6-keto-PGF,, values in the two groups were not signifi- cantly different (Figure I).

CPAR, platelet counts and blood viscosity CPAR showed no significant difference between control and anisodamine-treated groups at 12 or 24 h postbum (Figtrre 2), however during 2-3 days it became significantly higher in the treated than in the control group, indicating

Table I. Estimation of intravenous fluid for resuscitation of adult bum patients

Electrolyte- Colloid-containing Electrolyte-free

containing fluids’ fluids t water

lst24h 1 ml/kg/%TBSA 0.5 ml/kg/%TBSA 5-l O%GS(dextrose) in water 2000ml 2nd 24 h 0.5 ml/kg/%TBSA 0.25 ml/kg/%TBSA 5-l O%GS(dextrose) in water 2000 ml

‘Electrolyte-containing fluids include 0.9% normal saline and isotonic sodium bicarbonate. The ratio of normal saline to sodium bicarbonate, is2:l. Kolloid-containing fluids include plasma, whole blood and usually low molecular weight dextran.

Days postburn

Figure 2. Changes in platelet count, CPAR and blood viscosity. - Anisodamine-treated groups; -‘-.-, control group. *lJ< A.05; “PC 0.01.

that less circulating platelet aggregates were presented after the infusion of anisodamine. Also the platelet counts increased and were significantly higher at I, 2 and 3 days postbum in the treated than in the control groups. In contrast, blood viscosity was markedly lower in the treated than in the control groups at 2 and 3 days postbum.

Variables indicating tissue ischaemia, cellular destruc- tion and enzyme release CPK increased in both groups postbum, however in the control group it showed significantly higher levels and a longer duration of increase as compared with that in the treated group, and remained higher than normal (less than 90 units) even at day 7 postbum. Levels of LDH at 2, 3, 5 and 7 days postbum and levels of GOT at I and 2 days were significantly higher in controls than in the treated patients (Figure 3).

Plasma cortisol and 24 h urine epinephrine At 24 h postbum the plasma cortisol levels in both groups were at the same level; thereafter they declined gradually in the treated group to a 48 h nadir, whereas in the controls they increased further at these times before showing decreases 5 days later. The levels of epinephrine in 24 h urine samples were significantly higher at 3 and 5 days postbum in controls than in the treated group (Figure 4).

Discussion Roles of TXA, and PGI, imbalance in burn shock This study showed that both plasma TXA, levels and the

Huang et al.: Thromboxane and anisodamine in bum shock 251

Days postburn

Figure 3. Changes in CPK, LDH and GOT. - , Anisodamine- treated group; -.-.-, control group. “PC 0.05; ‘*PC 0.01.

TXA,/PGI, ratio increased in burned patients, with a significantly higher elevation during the early stages post- bum. In parallel with the patients’ recovery from shock these values concomitantly and gradually decreased, suggesting that TXA, played an important part in the genesis of bum shock. It has been reported that TXA, is an important mediator leading to early burn oedema, progressive dermal ischaemia, microcirculatory disturbances and increased microvascular permeability (DelBeccaro et al., 1980; Robson et al., 1980; Alexander et al., 1984; Heggers et al., 1985).

TXA, can directly constrict vascular smooth muscles and affect cardiovascular functions and indirectly cause cellular destruction, destabilize cell membranes and promote the release of various enzymes and some other bioactive substances from cells. The increased values of CPK, LDH and GOT in this study indicated that tissue ischaemia and cellular destruction had occurred. Moreover, significant correlations were found between TXA, and CPK, TXA, and LDH, and TXA, and GOT (rz0.796, 0.756 and 0.797 respectively, P< O.Ol), suggesting that tissue ischaemia, cellular destruction and enzyme release were closely related to the increased levels of TXA,. An additional interesting finding was observed at days 1-3 postbum when control values of CPK, LDH and GOT increased further and were much higher than those at 12 h postbum. This may be attributed to the release of tissue products following reperfusion of ischaemic tissue. It has been reported that increases of TXA, and TXA,/PGI, ratios are responsible for the reperfusion damage of ischaemic tissues (Lelcuk et al., 1985; Alemayehu et al., 1987; Anner et al., 1988; Kaufman et al., 1988). In this study the reperfusion damage following fluid resuscitation of bum shock can be attributed to increases in the TXA, content of the tissues.

A series of haemorheological changes may follow major bums among which platelet aggregation may be very important. The triggering of platelet aggregation by TXA, is an important factor in thrombus formation (Moncada and Vane, 1979). Mallarkey and Smith (1984) reported that TXA, is the most potent endogenous stimulus known for irreversible platelet aggregation, which induces platelet releases of ADP and 5-HT. Another effect of TXA, is to serve as a carrier of calcium which enhances the calcium concentrations in plasma and platelets and the activity of phospholipase A,, thereby promoting TXA, and other prostaglandin synthesis; hence a vicious circle is formed

10 6 0

I I I %\ I I 1 2 3 " 5 7

Days postburn

Figure 4. Changes in plasma cortisol and 24 h urine epinephrine. -, Anisodamine-treated group; -.-‘-, control. ‘PC 0.05; T< 0.01.

(Zhang and Wang, 1985). Our study showed that platelet counts decreased in the

controls within 5 days postbum and began to recover at day 7 postbum, whereas CPAR remained at low levels in the early stages postbum indicating more platelet aggregate formation. Further analysis showed that CPAR was linearly correlated with platelet count (r= 0.817, P< O.Ol), strongly suggesting that increased platelet microaggregate forma- tion may be one of the reasons for the decreased platelet counts. Blood viscosity also increased at these times and simultaneously TXA, and the TXAJPGI, ratio increased markedly in the control group. Regression and correlation analyses showed that TXA, was closely correlated with CPAR, platelet count and blood viscosity (r= -0.691, - 0.862 and 0.823 respectively, P < 0.05401). These results suggest that TXA, played a role in the haemorheolo- gical changes following severe bums. The chain of mech- anisms might be: microvascular constriction (Huang et al., 1987), leading to increased resistance to blood flow and circulatory stasis, the combining of TXA, receptors with the membranes of platelets (Kennedy et al., 1983), reductions in plasma CAMP levels affecting platelets and enhancing the plasma calcium levels leading to further platelet distortion, aggregation and release reactions (Whittle and Moncada, 1983).

Therapeutic roles and mechanisms of action of the TXA, synthesis inhibitor anisodamine in bum shock In order to confirm the roles of TXA, and PGI, imbalance in the genesis of bum shock, a TXA, synthesis-inhibitor- treated group was produced for this study. A number of drugs have been shown to inhibit TXA, synthesis. In vitro studies (Xue et al., 1982) indicated that anisodamine (a Chinese vasoactive drug extracted from ‘Anisodus tangu- ticus’, a variety of Da&a) has this effect. When given in large doses or infused too quickly it causes tachycardia and hypotension as well as inhibiting TXA, synthesis. Ani- sodamine has been widely used in the treatment of both haemorrhagic and septic shock in China for many years. Although its effective antishock treatment has been shown in a large number of clinical and experimental studies, it has seldom been used for the treatment of bum shock. In this study both TXA, and the TXA,/PGI, ratios were modified by the administration of anisodamine with significant

252 Bums (1990) Vol. 16/No. 4

reductions following treatment. In contrast there was no marked difference in the PGI, levels between the two groups. These findings suggested that anisodamine could inhibit TXA, synthesis in burned patients, but did not affect PGI, synthesis. .

The platelet counts and CPAR in the treated group were higher than in the controls, indicating that infusion of anisodamine reduced circulatory platelet aggregate forma- tion. This effect was, to a certain extent, attributed to inhibition of the influence of TXA, on haemorheology by anisodamine, and might be very important in limiting multiple organ failure in burned patients. Such failure resulting from microaggregate formation has been described as one of the major pathogenic pathways leading to multiple organ failure @hang, 1983). Our study also showed that the infusion of anisodamine improved tissue perfusion, lessened cellular and tissue ischaemia and destruc- tion and reduced the release of cellular enzymes.

After bums the peak value of CPK in the treated group was 1.94 times higher than the normal value (less than 90 units) and had returned to normal by 5 days postbum. However in the control group the peak value of CPK was 2.7 times higher than normal and remained as high as 98 units by day 7 postbum. Concomitantly the levels of LDH and GOT in the treated group were significantly lower than found in the controls. Furthermore the TXA, levels were highly correlated with CPK, LDH and GOT. These results confirmed that anisodamine lessened the effects of TXA, on tissue ischaemia, cellular destruction and enzyme release.

It was also found in this study that at l-3 days postburn CPK, LDH and GOT in the treated patients were only slightly increased as compared to those at 12 h postbum, suggesting that apparent reperfusion damage did not occur with the infusion of anisodamine. In addition anisodamine modified the levels of plasma cortisol (Figure 4) which began to decline 2 days after burning in the treated group, whereas they continued to increase in the controls. The epinephrine content in 24 h urine samples showed changes similar to those of cortisol, suggesting that anisodamine could enhance the restoration to normal of the neuroendocrine disturbance initiated by shock (stress). It is well known that cortisol and epinephrine are the main hormones released when the host is stressed, the extent of the hormone changes, to a certain degree, reflects the severity of the injury stimulus.

It is unclear exactly how the anisodamine modulated the beneficial effects. This study suggests that it may have been due to the inhibition of the harmful effects of TXA, which improved tissue perfusion, reduced cellular destruction and the release of bioactive substances. These changes improved the general condition of the patients with return of the neuroendocrine disturbances to normal and shown by a decreased secretion of cortisol and epinephrine.

In conclusion, this study showed an imbalance of TXA,/PGI, and a protective effect of a TXA, synthesis inhibiitor, anisodamine, in bum shock. Increased TX.4, played an important role in changes of haemodynamics and haemorrheology. Anisodamine inhibited TXA, synthesis and reduced platelet aggregation and microthrombus for- mation. Moreover, treatment with anisodamine decreased the plasma levels of CPK, LDH and GOT and the secretion of cortisol and epinephrine. These beneficial effects could be mediated by inhibition of vasoconstriction, platelet aggre- gation and cellular destruction and the destabilization of cell membranes which are induced by TXA,, thus maintaining tissue perfusion and preserving cellular integrity and result-

ing in reduced release of lysosomal enzymes. Our study also showed that TX& was responsible for the reperfusion damage after fluid infusion and that anisodamine could reduce this damage. These results support the role of TXA, as an important mediator of bum shock and suggest that inhibition of TXA, synthesis may be useful in the treatment of patients with bum shock.

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prostacyclin and thromboxane production in irreversible hemorrhagic shock. Circ. Shock 23, 119.

Alexander F., Mathieson M. Keoh H. et al. (1984) Arachidonic acid metabolites mediate early bum edema. 1. Truuma 24, 709.

Anner H., Kaufman R. P., Valeri C. R. et al. (1988) Reperfusion of ischemic lower limbs increases pulmonary microvascular permeability. 1. Trauma 28,607.

Bitterman H., Yanagisawa A. and Lefer A. M. (1986) Beneficial actions of thromboxane receptor antagonism in hemorrhagic shock. Circ. Shock 20,l.

Bum Research Unit, Third Military Medical College (ed.) (1977) Treafment of Bums, 1st edn. Beijing: People’s Health Press, pp. 108-109.

Carmona R. H., Tsao T. C. and Trunkey D. D. (1984) The role of prostacyclii and thromboxane in sepsis and septic shock. Arch. Surg. 119,189.

DelBeccaro E. J., Robson M. C., Heggers J. P. et al. (1980) The use of specific thromboxane inhibitors to preserve the dermal microcirculation after burning. Surgery 87, 137.

Heggers J. P., Robson M. C. and Zachary L. S. (1985) Throm- boxane inhibitors for the prevention of progressive dermal ischemia due to the thermal injury. J. Bum Care Rehabil. 6,466.

Huang Y. S., Li Ao and Yang Z. C. (1987) Roles of thromboxane and prostacyclin in pulmonary diseases. Fureim Med. (on respiratory system). 7, 15.

Kaufman R. P., Klausner J. M., Anner H. et al. (1988) Inhibition of thromboxane (TX) synthesis by free radical scavengers. 1. Trauma 28, 458.

Kennedy I., Coleman R. A., Humphrey P. P. et al. (1983) Studies on the characterization of prostanoid receptors. In: Samuelsson B. et al. (eds), Advances in Prostaglundins, ‘Thromboxane and Larks- friene Research, vol. 11. New York: Raven, p. 327.

Lelcuk S., Alexander F., Kobzik L. et al. (1985) Prostacyclin and thromboxane A2 moderate postischemic renal failure. Surgery 98, 207.

Li Z. J., Yang M. F., Hao X. H. et al. (1985) Radioimmunoassay for thromboxane B, in human plasma. Med. 1. Chin. PLA 10,35.

Mallarkey G. and Smith G. M. (1984). The effect of arachidonic acid metabolism on intravascular platelet aggregation in rats. 7hromb. Res. 36, 91.

Moncada S. and Vane J. R. (1979) Arachidonic acid metabolites and the interactions between platelets and blood-vessel walls. N. Engl. 1. Med. 300, 1142.

Reines H. D., Halushka P. V., Cook H. A. et al. (1982) Plasma thromboxane concentrations are raised in patients dying with septic shock. Lancet ii, 174.

Robson M. C., DelBeccaro E. J., Heggers J. P. et al. (1980) Increasing dermal perfusion after burning by decreasing thromboxane production. 1. Trauma 20, 722.

Whittle B. J. R. and Moncada S. (1983) Pharmacological inter- actions between prostacyclin and thromboxanes. Br. Med. Bull. 39, 232.

Huang et al.: Thromboxane and anisodamine in bum shock 253

Wu K. K. and Hoak J. C. (1974) A new method for the quintitative detection of platelet aggregates in patients with arterial insufficiency. Lancef ii, 294.

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Xue R. J., Hammerschmidt D. E., Coppo P. A. et al. (1982) Anisodamine inhibits thromboxane synthesis, granulocyte aggregation, and platelet aggregation. ]AMA 247, 1458.

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Paper accepted 6 December 1989.

Correspondence shouti be addressed to: Professor Li Ao (Ngao), Bum Institute, Southwestern Hospital, Third Military Medical College of PLA, Chongqing, Sichuan, China.

First International Conference on Bums and Fire Disasters

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For technical reasons it has been found necessary to bring the date of the Meeting one week forward from 2-5 October 1990 to 25-28 September 1990

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